PROJECT SUMMARY Human Cytomegalovirus (HCMV) is a ?-herpesvirus that establishes life-long infection in over 60% of the world population. While innocuous in most healthy individuals, HCMV is the leading infectious cause of congenital birth defects ranging from hearing or vision loss and cognitive impairment, to severe developmental disabilities, microcephaly and death. Yet awareness of this in the general public is alarmingly low, leading many experts to refer to HCMV as the ?silent global burden?. In adults, HCMV is a leading cause of restenosis and coronary problems, has been linked to some cancers, and causes major complications in immunosuppressed transplant recipients or AIDS patients. Despite this, there is no vaccine or cure, and we continue to have a relatively limited understanding of HCMV replication when compared against other viruses. Indeed, unlike most other viruses, HCMV has a protracted replication cycle spanning several days during which time it forms a unique cytoplasmic site for virion maturation, termed the Assembly Compartment (AC). While recent fixed imaging approaches have provided insights into its structure revealing that it comprises a remodeled Golgi surrounded by various host organelles and vesicles, a detailed mechanistic understanding of HCMV replication has been limited in large part by the challenges associated with imaging the AC and host organelles over extended periods in living cells. In preliminary data supporting this proposal, we develop innovative new multi-color live cell imaging approaches that provide the first insights into AC and infected cell behavior, resulting in the unexpected finding that the AC acts as a novel virus-assembled microtubule organizing center (MTOC) that enables HCMV to rotate the host cell nucleus in preparation for cell migration. HCMV accomplishes this by directly targeting and recruiting the highly specialized microtubule (MT) end-binding protein, EB3 to the AC. This serves to recruit specific EB3-associated regulators of MT nucleation and polymerization, and allows the AC to generate specialized subsets of MTs that are heavily acetylated. Acetylation of AC-derived MTs confers the mechanical strength needed to rotate host nuclei and control cell adhesion and migration, which promote virus spread. Our data also shows that RNA interference (RNAi)-mediated suppression of EB3 expression blocks nuclear rotation and suppresses virus spread. Independently, we develop a small myristoylated peptide that is rapidly taken up by primary cells and interferes with EB3-dependent recruitment of host proteins to the AC. This peptide, but not control peptides, blocks HCMV-induced rotation of the nucleus and acts as a non- toxic, virus-specific inhibitor of HCMV spread. In this proposal, we will test several hypotheses and alternatives to determine the mechanistic details of how the AC functions as a novel MTOC through control of EB3, and determine both how and why HCMV induces rotation of the host cell nucleus when promoting cell migration and virus spread. Accomplishing these Aims will not only provide innovative new tools for research alongside mechanistic new insights into HCMV replication in living cells, but also has significant translational potential.